Since the extracellular cyst wall is required for transmission of giardiasis, the overall goal of this project is to gain an in-depth understanding of the structure and regulation of the major cyst wall proteins and their processing and pathways of transport. Dr. Gillin has completed the life cycle of G. lamblia in vitro for the first time and shown that encystation entails expression of two groups of regulated antigens and formation of encystation- specific secretory vesicles (ESV) that deliver them to the cell surface. Monoclonal antibody GCSA-l recognizes Group I proteins of 26, 32, and 44 kilodalton (kD), which are expressed early in encystation. However, only the 32 kD species is recovered from cyst walls. In contrast, Group 2 consists of four WGA-reactive cyst wall glycoproteins (66-140 kD) that are expressed after 19 hours of encystation. Antibodies against purified gpl20 recognize, protein epitopes of both major regulated proteins (120 and 85 kD). The investigator will now build on these important findings to delineate key molecular biochemical and cellular pathways of cyst wall formation. Specific Aim A is to trace the origin, formation, and traffic of the ESV contents and membrane by sophisticated light and electron microscopic techniques and to ask whether the two groups of cyst wall antigens follow the same pathways of transport and release. Specific Aim B is to compare the kinetics of expression, processing and release of these cyst wall proteins and glycoproteins by quantitative analyses of pulse-chase labeling and immunoprecipitation with anti GCSA-l or anti-gp 120/85. Chemical analyses will reveal the proportion of N- and O-glycosylation and identify the sugars of isolated gpl20 and gp85. In Specific Aim C, anti-GCSA-l and anti-gp 120/85 will be used to isolate the genes that encode a GCSA-l gene and either gpl20 or gp85 from their expression library. The sequence, copy number, expression during encystation, and conservation of these genes will be analyzed. Sequences of the major cyst wall protein and glycoprotein will give important insights into their structures and functions. In Specific Aim D, encysting cells will be fractionated in order to isolate the ESV and determine whether they contain enzyme(s) of cyst wall biosynthesis, that she has described. Moreover, since she has found that disulfide bonds are important in the architecture of the cyst wall, she will determine where these bonds form. These studies will greatly enhance our understanding of giardia encystation and cyst wall formation and could point the way to interruption of this key process, in the future.